I propose to investigate the actin bundles of three biological systems chosen as models to help us understand how actin filaments produce motility in non-muscle cells. The first of the bundles is found in the sperm of Limulus (horseshoe crab); the second in microvilli; and the third in the sperm of Mytilus (mussel). Bundles of actin filaments are common features of almost all eukaryotic cells and often are involved in motility. Both the bundles in the sperm of the horseshoe crab and the mussel are motile structures. During fertilization of the egg, the bundles are explosively pushed outward to form the long, slender acrosomal process. The polarity of the actin filaments in the bundle is such that the motion could not be generated by myosin. Therefore, I expect to uncover new mechanisms of actin-based motility from these studies. Indeed, based on detailed studies in the first three years of this grant, there is good evidence that the extension of the Limulus bundle is driven by a heretofore undescribed mechanism; namely, a change in twist of the component actin filaments. Because the Limulus bundle exemplifies a new dynamic property of actin, I plan to extend there studies in order to understand the construction and assembly of this bundle during spermiogenesis. It is likely that actin bundles in other structures beside that in Limulus also have the capability to change twist and generate motion. I think the bundle of actin filaments forming the core of the microvillus may rotate as a result of a change in twist of the filaments. While such rotation could not be seen in the light microscope, in the electron microscope I can study the supercoiling of the bundle which is a structural consequence of such rotation. Another motile bundle is that found in the sperm of Mytilus. Based on what is known, it seems impossible that the extension is a result of any of the known mechanisms: i.e., an interaction of actin and myosin, a change in twist of the filaments in the bundle, or elongation by polymerization of actin subunits. Therefore, by examining the detailed structure of the sperm before and after the extension, I expect to uncover a wholly new dynamic property of actin.